1,721,185 research outputs found
The mitochondrial outer membrane protein assembly machinery of the human fungal pathogen Candida albicans
No full textMitochondria are essential organelles with protein complexes embedded in their membranes. These act as molecular machines that produce energy and carefully regulate what goes in to and out of the organelle. The outer membrane machinery provides the interface between the mitochondrion and the rest of the cell and coordinates the activity of each mitochondrion in keeping with the metabolic requirements of the cell. Research over the last twenty years has identified many of the components and complexes that assemble this membrane machinery, but many questions remain about how these machines function and how they are assembled. While we have learnt a great deal about these molecular machines in the tractable model system Saccharomyces cerevisiae, its unusual regulation of mitochondrial function makes it difficult to glean general principles from discoveries in this organism alone. By comparing findings in other organisms we can determine which features are conserved and learn more about the evolutionary origins of the molecular components. The work in this thesis focuses on the protein import machinery of the mitochondrial outer membrane, in particular the sorting and assembly machinery (SAM) complex. My thesis is the first in-depth study of the mitochondrial import machinery of the human fungal pathogen Candida albicans. I outline the mitochondrial protein import pathways with a focus on the roles and partnerships of the SAM complex. Examining the import pathways in C. albicans in comparison to S. cerevisiae revealed interesting examples of rewiring of the protein import pathways into the intermembrane space. These preliminary studies also show that mitochondria from C. albicans more efficiently assembles proteins into the mitochondrial outer membrane, which leads to a more detailed investigation of the composition and behaviour of the SAM complex. This reveals new roles for the SAM complex components; Sam37, Sam35 and Mdm10 and a new component of the C. albicans SAM complex, Sam51. The work presented here describes the functions and interactions of C. albicans SAM complex components, a detailed characterisation of their roles in the import and assembly of outer membrane proteins, and the partnerships between the C. albicans SAM complex and other outer membrane protein complexes
Omp85 family proteins and the BAM complex in Caulobacter crescentus.
No full textThe outer membrane of Gram-negative bacteria is a compartment that houses many proteins involved in basic physiological functions, virulence and multi-drug resistance, and is therefore important for cell survival. Most proteins in the outer membrane adopt a β-barrel conformation, and require the β-barrel assembly machinery (BAM) for integration into the outer membrane. The BAM complex is a multi-subunit protein complex present in the outer membrane of all Gram-negative bacteria. It comprises of a core β-barrel protein, BamA and associated lipoproteins that collectively participate in the folding and insertion of β-barrel proteins. BamA has functional homologues in eukaryotes that form the SAM complex in mitochondria for insertion and assembly of β-barrel proteins into the mitochondrial outer membranes. Previous work by other groups in the field has used N. meningitidis (β-proteobacteria) and E. coli (y-proteobacteria) to study the function of BamA and the BAM complex. In this comparative study, we will use Caulobacter crescentus as a model α-proteobacterium to learn about differences and similarities in the BAM complexes across the three different classes of proteobacteria. The combined use of bioinformatics and biochemical experiments in C. crescentus have shown some key differences with the absence of the BamC partner lipoprotein and the presence of a novel OmpA-like protein as well as two other uncharacterized lipoprotein partners for BamA. In Chapter 2, we perform a comparative analysis of BamA and the BamA-like protein (Omp68) in C. crescentus. The Omp85 superfamily contains BamA, Omp68 and TpsB proteins and using bioinformatics analysis, we proposed that Omp68 is an evolutionary intermediate of BamA and TpsB proteins. We first established a method for preparing outer membranes from C. crescentus and demonstrated that like BamA, Omp68 forms an oligomeric complex in the outer membrane although it has characteristics that could classify it as a TpsB protein. The unusual nature of Omp68 is discussed and its possible functions. A method for effective purification of the BAM complex from outer membranes was also established for identification and analysis of the different components of the complex. In Chapter 3, we demonstrate that the BAM complex possesses modular characteristics and contains BamA and six outer membrane lipoproteins in the α-proteobacterium, C. crescentus. In addition to the three known lipoproteins (BamB, BamD and BamE), we identify three other subunits (Pal, BamF and BamG) of which only Pal is essential. We propose that Pal is a protein that anchors the BAM complex to the peptidoglycan layer and promotes proximity to the inner membrane Sec machinery for efficient outer membrane protein assembly. We also show BamF is a genuine component of the BAM complex and a potential homologue of the BamC protein found in all other proteobacteria. Both BamF and BamC contain a conserved motif that is possibly important for docking onto the BAM complex. In Chapter 4, we focus on biochemical and structural characterisations of BamD from C. crescentus. We show BamD is an outer membrane lipoprotein that forms the halo module of the BAM complex. We also demonstrate BamD contains TPR motifs that are essential for functioning of the BAM complex and that protrude into the periplasm for protein-protein interactions. Further experiments involved extensive optimisations of heterologous expression of BamD and purification with the aim of progressing to crystallisation trials. We were able to generate purified folded BamD that will be used for future experiments
Assembly of multimeric outer membrane proteins in Escherichia coli
No full textGram-negative bacterial outer membranes incorporate proteins of at least three well characterized architectures: β-barrel proteins, lipoproteins and multimeric secretion pores. Assembly of β-barrel proteins into the outer membrane is mediated by the β-barrel assembly machinery (BAM), consisting of an essential core β-barrel BamA and four accessory lipoproteins (BamBCDE). Lipoproteins are targeted and subsequently anchored to the outer membrane by covalently attached lipid modifications by the localization of lipoproteins (Lol) machinery. Secretins are amongst the most characterized multimeric outer membrane proteins, and often rely on a small lipoprotein, termed pilotin, to catalyze the efficient translocation of secretin monomers to the outer membrane for assembly into a functional secretion pore. Through the use of cryo-EM and X-ray crystallography it has become clear that oligomeric outer membrane proteins can adopt either α-helical or β-stranded transmembrane conformations, yet their mechanism of assembly remains unclear. Chapter two describes the identification and characterisation of a novel pilotin AspS for the secretin GspD from the type 2 secretion system of enteropathogenic Escherichia coli. Sucrose density fractionation and novel time course assays show that AspS is required for the targeting of GspD to the outer membrane and for the efficient assembly of GspD multimers. The structure of AspS was solved, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to pilotins from other secretion syetems. Chapter three describes the optimization of a bamA depletion strain of E. coli to monitor outer membrane protein assembly. Assays were performed to analyse bacterial viability, morphology and functionality of the BAM after bamA depletion. Chapter four focusses on analysing the role of BamA in assembling secretion channels GspD, Wza and CsgG, using the depletion regime described in Chapter three. Assembly assays demonstrate that GspD, Wza and CsgG can assemble into the outer membrane in the absence of BamA. These assays also showed that periplasmic proteins CsgE and CsgF could enhance the assembly efficiency of both the mature CsgG multimer and a possible CsgG assembly intermediate
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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